The nature of this grant is to investigate ontogenetically and phylogenetically the mechanism(s) of adaptive immunity in ectothermic vertebrates and advanced invertebrates. Application of in vitro mitogen models to in vivo test systems in leopard frogs will be used to define the role of temperature in regulating and modulating adaptive immunity. The test system will include: 1) the use of T cell independent and dependent antigens in conjunction with temperature manipulations, 2) carrier-hapten immunization studies and temperature manipulations to determine whether T-helper cells are temperature sensitive, and 3) further studies on the enhancement of humoral and cellular immune responses as a result of low temperature. Mitogen studies on turtle lymphocytes will be investigated with an emphasis on activation kinetics, cell surface receptors for mitogens, specificity of responses and temperature differential responses. A comparison of mitogen cell surface receptors (i.e., sugar inhibition studies) in mice with those of frogs will be examined to determine whether differences exist between these two phylogenetic levels. The relationship between the stage of amphibian development or age and lymphocyte function with respect to particular lymphoid organs will be examined in frogs. The immunological aspects of the bone marrow compartment in bullfrogs will be investigated with respect to: 1) cellularity, 2) lymphopoiesis and 3) immune response. The role of temperature, the ontogeny of mitogen responsiveness, the nature of lymphocyte polyclonal expansion and a comparison of amphibian immunity with that of mammals will either confirm or disprove the phylogenetic conservation of these events during the evolutionary development of vertebrate immunity. Using earthworm coelomocytes, we plan to focus primarily on in vitro work concerning phagocytosis and cell division. These studies will continue to emphasize the universality of immune response, supporting the hypothesis that cell-mediated and humoral immunity underwent progressive evolution during development.